NME1 Expression Research Articles

Transcriptome analysis of adherens junction pathway-related genes after peripheral nerve injury.

The neural regeneration process is driven by a wide range of molecules and pathways. Adherens junctions are critical cellular junctions for the integrity of peripheral nerves. However, few studies have systematically characterized the transcript changes in the adherens junction pathway following injury. In this study, a rat model of sciatic nerve crush injury was established by forceps. Deep sequencing data were analyzed using comprehensive transcriptome analysis at 0, 1, 4, 7, and 14 days after injury. Results showed that most individual molecules in the adherens junctions were either upregulated or downregulated after nerve injury. The mRNA expression of ARPC1B, ARPC3, TUBA8, TUBA1C, CTNNA2, ACTN3, MET, HGF, NME1 and ARF6, which are involved in the adherens junction pathway and in remodeling of adherens junctions, was analyzed using quantitative real-time polymerase chain reaction. Most of these genes were upregulated in the sciatic nerve stump following peripheral nerve injury, except for CTNNA2, which was downregulated. Our findings reveal the dynamic changes of key molecules in adherens junctions and in remodeling of adherens junctions. These key genes provide a reference for the selection of clinical therapeutic targets for peripheral nerve injury.

Abstract 5536: The metastasis suppressor NME1 regulates stemness in melanoma

Abstract Melanoma is a lethal skin cancer that is highly resistant to therapy and prone to metastasize to distant organs. The quiescence melanoma stem cells are believed to account for the resistance to therapies. Traditional chemotherapies designed to target fast-proliferative tumor cells tend to miss quiescent stem cells. The escaped quiescent stem cells may accumulate oncogenic mutation from common mutagenic sources and finally lead to tumor relapse. NME1 is a metastasis suppressor gene that inhibits the metastatic potential in melanoma and other cancers. In this study, we focus on the role of NME1 in the regulation of stemness and quiescence of melanoma stem cells. We find that the genesis and growth of stem cell spheroids derived from melanoma cell lines are abrogated after knockdown of NME1. We also find that the mRNA level of several melanoma stem cell markers are elevated when we knockdown NME1. These results suggest that NME1 regulates the survival and stemness of melanoma stem cells. In addition, we demonstrate that melanoma stem cells are heterogeneous in NME1 expression. Specifically, the NME1 expression is markedly elevated in a highly proliferative subpopulation in the spheroids that express the stem cell markers OCT4, SOX2 and SOX10. Conversely, we observe another subpopulation of these spheroids that exhibits low NME1 expression and is non-proliferative. We hypothesize that this subpopulation consists quiescent melanoma stem cells that are more resistance to therapies, and that their quiescent status is controlled by the level of NME1. We are now employing CRISPR technology to separately study potentials in tumorigenesis, metastasis, and therapy-resistance of the stem cell subpopulations. Citation Format: Ying Wang, David M. Kaetzel. The metastasis suppressor NME1 regulates stemness in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5536. doi:10.1158/1538-7445.AM2017-5536

Abstract 4845: Induction of aldolase C expression by NME1 may mediate metastasis suppressor activity in malignant melanoma

Abstract Although the metastasis suppressor activity of NME1 has been demonstrated in a number of settings of human cancer, the underlying molecular mechanisms are not well understood. NME1 has two known enzymatic activities, nucleoside diphosphate kinase (NDPK) and a 3’ to 5’ exonuclease. To determine the relative contribution either of these activities has on the metastasis suppressor function of NME1, our lab previously generated site directed mutants to abrogate one or both of the enzymatic activities (Ma et al., 2004). These studies showed that disrupting the 3’ to 5’ exonuclease activity with the point substitutions glutamate5-to-alanine (E5A) and lysine12-to-glutamine (K12Q) also significantly compromised the metastasis suppressor function of NME1 (Zhang et al., 2011). To identify genes that are regulated by NME1 and potentially modulate its metastasis suppressor activity, we compared the gene expression profile induced by forced expression of wild-type NME to those obtained with the E5A and K12Q mutants. Using the human Exon 1.0 Affymetrix microarray , we identified 13 genes whose expression was modulated by wild-type NME1 expression, but not by the suppressor-deficient mutants, which were designated as metastasis suppressor signature (MSS) genes. We are currently refining the list to genes that have the highest clinical relevance through observing expression profiles of the MSS genes in clinical datasets. Preliminary results of unsupervised hierarchal clustering of GEO melanoma dataset GSE8401 showed that expression of the MSS gene, aldolase C (ALDOC), was significantly lower in a subset of metastatic melanoma lesions compared to primary melanomas. Western blot and qPCR analysis confirm that NME1 induces expression of ALDOC in both WM793 and M14 melanoma cell lines, and that induction of ALDOC is dose-responsive to NME1 expression as observed in the WM1158 metastatic melanoma cell line. Further, NME1 induction of ALDOC is specific compared to the ALDOA and ALDOB isoforms. Recent studies have indicated aldolase isoforms can modulate motile and invasive phenotypes of cancer cells (Li et al., 2015 & Hu et al., 2016), suggesting regulation of ALDOC expression may represent a key mechanism underlying the metastasis suppressor function of NME1. Current work is focused on measuring the impact of NME1-induced expression of ALDOC on the metastatic potential of melanoma cells in culture and in vivo. Citation Format: Nidhi Pamidimukkala, Katie Leonard, Joseph McCorkle, Qingbei Zhang, Anup Mahurkar, Amol Shetty, David Kaetzel. Induction of aldolase C expression by NME1 may mediate metastasis suppressor activity in malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4845. doi:10.1158/1538-7445.AM2017-4845

Abstract 4847: NME1 mediates a switch in beta integrin subunits that correlates with prolonged patient survival

Abstract Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility, invasion, and metastasis, but the molecular mechanisms underlying this activity are not completely understood. Herein we report a novel mechanism through which NME1 modulates focal adhesion dynamics via regulation of integrins β1 and β3. Stable expression of NME1 significantly altered focal adhesion turnover at the cell periphery. Interestingly, over-expression of NME1 resulted in a switch from predominantly fast recycling α4β1 integrins to slower recycling αvβ3 integrins. Contrary to its regulation of other cell surface receptors, the inhibition of integrin β1 and induction of integrin β3 by NME1 was found to occur at the transcriptional level rather than through dynamin mediated endocytosis. Induction of integrin β3 required both the 3-5’ exonuclease and nucleoside diphosphate kinase (NDPK) activities of NME1, which are also required for its metastasis suppressor activities in vivo. Further suggesting that the induction of integrin β3 is involved in the metastasis suppression function of NME1, knockdown of integrin β3 significantly increased the invasion capability of cells expressing NME1 in vitro compared to control cells. Analysis of metastatic melanoma patients in the TCGA showed individuals with a 1.5x or greater increase in integrin β3 mRNA had a significantly longer overall survival times. Additionally, an inverse correlation was observed between NME1 and integrin β1 mRNA in an independent microarray of primary melanomas. The inverse correlation of NME1 and integrin β1 RNA was also a strong predictor of prolonged distant disease free and overall survival in patients with the basal-like subtype of breast carcinoma. Together, these data strongly suggest NME1 prevents metastasis of human melanoma and some types of breast cancers by altering beta integrin expression to reduce recycling of focal adhesions and, ultimately suppress of cell motility. Citation Format: M. Kathryn Leonard, Grace Snow, Marián Novak, Joseph R. McCorkle, Xiuwei H. Yang, Alexey Belkin, David M. Kaetzel. NME1 mediates a switch in beta integrin subunits that correlates with prolonged patient survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4847. doi:10.1158/1538-7445.AM2017-4847

Prognostic Value of NME1 (NM23-H1) in Patients with Digestive System Neoplasms: A Systematic Review and Meta-Analysis.

ObjectiveThere is a heated debate on whether the prognostic value of NME1 is favorable or unfavorable. Thus, we carried out a meta-analysis to evaluate the relationship between NME1 expression and the prognosis of patients with digestive system neoplasms.MethodsWe searched PubMed, EMBASE and Web of Science for relevant articles. The pooled odd ratios (ORs) and corresponding 95%CI were calculated to evaluate the prognostic value of NME1 expression in patients with digestive system neoplasms, and the association between NME1 expression and clinicopathological factors. We also performed subgroup analyses to find out the source of heterogeneity.Results2904 patients were pooled from 28 available studies in total. Neither the incorporative OR combined by 17 studies with overall survival (OR = 0.65, 95%CI:0.41–1.03, P = 0.07) nor the pooled OR with disease-free survival (OR = 0.75, 95%CI:0.17–3.36, P = 0.71) in statistics showed any significance. Although we couldn’t find any significance in TNM stage (OR = 0.78, 95%CI:0.44–1.36, P = 0.38), elevated NME1 expression was related to well tumor differentiation (OR = 0.59, 95%CI:0.47–0.73, P<0.00001), negative N status (OR = 0.54, 95%CI:0.36–0.82, P = 0.003) and Dukes’ stage (OR = 0.43, 95%CI:0.24–0.77, P = 0.004). And in the subgroup analyses, we only find the “years” which might be the source of heterogeneity of overall survival in gastric cancer.ConclusionsThe results showed that statistically significant association was found between NME1 expression and the tumor differentiation, N status and Dukes’ stage of patients with digestive system cancers, while no significance was found in overall survival, disease-free survival and TNM stage. More and further researches should be conducted to reveal the prognostic value of NME1.

Wortmannin as Targeted Therapeutic Agent for the Treatment of Triple-Negative Breast Cancer

Purpose: To investigate the inhibitory effect of wortmannin on inhibition of BT-20 and BT-474 breast cancer cells in Athymic nu/nu mice model.Methods: Forty Athymic nu/nu mice were randomly assigned to 4 groups of 10 each, namely, BT-20 control, BT-20 treatment, BT-474 control and BT-474 treatment groups. The mice were injected with 2.5 x 105 BT-20 and BT-474 cells under anesthesia. Those in the treatment groups were given wortmannin (7 mg/kg) in DMSO daily intraperitoneally whereas the animals in control groups received an equal volume of DMSO for 21 days after the cancer attained palpable stage. Western blot analysis was carried out using enhanced chemiluminescence reagent while Protean IEF cell unit was used for 1-D electrophoresis.Results: The results showed a significant decrease in the growth of the tested cancer cell lines on treatment with wortmannin at 7 mg/kg daily for 21 days. The volume of tumor in the treatment group was reduced to 42.72 ± 9.45 compared to 79.43 ± 17.11 mm3 in the control group after 21 days treatment (p &lt; 0.005). It also changed the expression of microRNA in BT-20 cells. Exposure of BT-20 cells to wortmannin for 24 h resulted in the altered expression of proteomes. Wortmannin treatment increased the expression of miRs including miR 19a/b, 22, 29b/c, 181c/d, 195 and 663. Western blot data showed increase in the expression of NME1 (NM23 H1) and reduction in the expression of vimentin after treatment with wortmannin.Conclusion: Wortmannin can be of benefit in the treatment of human breast cancer.Keywords: Wortmannin, Vimentin, Tumor growth, Expression of microRNA, Proteomes

Abstract 3257: NME1 mediates a switch in expression of beta integrin subunits that correlates with prolonged patient survival

Abstract Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility and invasion in vitro and metastasis in vivo, but the molecular mechanisms underlying this activity are not completely understood. Herein we report a novel mechanism through which NME1 modulates focal adhesion dynamics via regulation of integrin β1. Stable expression of NME1 significantly altered focal adhesion turnover at the cell periphery. Interestingly, over-expression of NME1 resulted in a switch in from predominantly fast recycling α4β1 integrins to slower recycling αvβ3 integrins. Contrary to its regulation of other cell surface receptors, the inhibition of integrin β1 by NME1 was found to occur at the transcriptional level rather than through dynamin-mediated endocytosis. This effect of NME1 required both its 3-5′ exonuclease and nucleoside diphosphate kinase (NDPK) activities, which are also required for its metastasis suppressor activities in vivo. Furthermore, an inverse correlation was observed between NME1 and integrin β1 mRNA in a large cohort of primary melanoma biopsies. The inverse correlation of NME1 and integrin β1 RNA was also a strong predictor of prolonged distant disease-free and overall survival in patients with the basal-like subtype of breast carcinoma. Together, these data strongly suggest NME1 suppresses metastasis of human melanoma and some types of breast cancers by inhibiting integrin β1 expression, which reduces recycling of focal adhesions and suppresses cell motility. Citation Format: M. Kathryn Leonard, Marian Novak, Xiuwei Yang, Alexey Belkin, David Kaetzel. NME1 mediates a switch in expression of beta integrin subunits that correlates with prolonged patient survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3257. doi:10.1158/1538-7445.AM2015-3257

Metastasis suppressor NME1 regulates melanoma cell morphology, self-adhesion and motility via induction of fibronectin expression.

Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility and invasion in vitro and metastasis in vivo, but the underlying molecular mechanisms are not completely understood. Herein, we report a novel mechanism through which NME1 controls melanoma cell morphology via upregulation of the extracellular matrix (ECM) protein fibronectin. Expression of NME1 strongly suppressed cell motility in melanoma cell lines 1205LU and M14. The resulting sedentary phenotype was associated with a more flattened appearance and marked increases in actin stress fibre and focal adhesion formation. NME1‐induced focal adhesions were colocalized with dense deposits of fibronectin, which were absent or minimal in the corresponding NME1‐deficient parental lines. NME1 was a strong inducer of fibronectin mRNA and protein expression, shown with reciprocal approaches of forced NME1 expression and shRNA‐mediated knock‐down. Increased synthesis and ECM deposition of fibronectin was necessary for NME1‐induced cell spreading, as knock‐down of fibronectin opposed the effects of NME1 on cell morphology. Fibronectin knock‐down also reversed the ability of NME1 to promote aggregation when cells were plated on a non‐adherent substratum. Similarly, inhibiting activation of the fibronectin receptor integrin α4β1 with an anti‐α4 antibody reversed the motility‐suppressing effect of NME1. A positive correlation was observed between NME1 and fibronectin mRNA in clinical biopsies of normal skin, benign nevi and primary melanomas, but not in metastatic forms, suggesting the NME1/fibronectin axis represents a barrier to melanoma progression. In summary, these findings indicate fibronectin is an important effector of the motility‐suppressing function of NME1 in melanoma cells.

Microrna Expression and Drug-Induced Changes in Gene Expression Correlate with Ara-C Chemosensitivity in AML Cell Lines

Abstract:Acute myeloid leukemia (AML) is a heterogeneous disorder, which is characterized by chromosomal abnormalities and genetic alterations. Cytarabine (Ara-C) is the most commonly used nucleoside analog for treatment of AML. However, the use of Ara-C is associated with two important clinical complications namely, inter-patient variability in response and development of intrinsic resistance. The inter-patient variability in response can be partly associated with polymorphisms in proteins that are required for intracellular uptake and activation of Ara-C to its phosphorylated form. Apart from genetic polymorphisms, expression of proteins involved in the uptake, activation, and inactivation of Ara-C have been shown to correlate with the overall patient survival. Over the past decade, various studies have identified microRNAs as important post-transcriptional regulators of gene expression. However, there are no studies till date that have identified key miRNAs involved in regulation of Ara-C pathway genes. Identification of these miRNAs will help in targeting these miRNAs to further understand inter-patient differences in gene expression. Additionally, drugs can also influence gene expression. However, there is critical gap in literature regarding role of Ara-C in inducing changes in gene expression. Understanding the dynamics of gene expression due to miRNAs and drug-induced changes would help open new opportunities for development of improved treatment strategies.Thus, the objective of this study is to understand the role of microRNAs in altering cytarabine cytotoxicity by influencing expression of pharmacokinetics (PK) and pharmacodynamics (PD) genes (n=18) in AML cell lines representing different risk groups. We evaluated genome-wide miRNA expression in 7 AML cell lines from different risk groups (favorable risk group: Kasumi-1, ME-1; intermediate risk group: AML-193, KG-1; adverse risk group: HL-60, MV-4-11, MOLM-16). We also evaluated the impact of cytarabine-induced gene expression changes in these AML cell lines. The gene expression changes were correlated with the in vitro chemosensitivity.Our preliminary results indicate that there was a significant correlation between the baseline miRNA expression for 16 miRNAs and Ara-C IC50 values (selected shown in Figure 1). We also observed that 57 microRNAs were associated with gene expression levels of the selected 18 Ara-C pharmacogenes (selected shown in Figure 1). Four miRNAs (miR-425-5p, miR-517a-3p, miR-519b-5p+hsa-miR-519c-5p, miR-522-3p) were found to be significantly associated with both gene expression and Ara-C IC50 values. We found that there were significant changes in gene expression of Ara-C pathway genes following treatment with 1uM or 10uM Ara-C. Briefly, there were significant changes in DCK, SLC29A1, CTPS1, CMPK1, NME1 and XRCC1 expression when treated with 10uM Ara-C and RRM2, NME1 and XRCC1 expression when treated with 1uM Ara-C.In conclusion, drug-induced changes in gene expression and miRNAs expression were found to correlate with chemosensitivity of AML cell lines. The preliminary results from our study help provide an insight into potential/additional molecular mechanisms associated with resistance observed in AML patients. Such knowledge is clinically significant, as identification of factors that contribute to the variable drug response would help in understanding and thus improving the variability in efficacy associated with cytarabine therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dual functions of NME1 in suppression of cell motility and enhancement of genomic stability in melanoma

The NME1 gene represents the prototypical metastasis suppressor, whose expression inhibits cell motility and metastasis without impact on primary tumor growth in a number of different human cancers. This report outlines our recent efforts to define the molecular mechanisms through which NME1 both suppresses cell motility and promotes genomic integrity in the setting of human melanoma. Forced NME1 expression in a variety of melanoma-derived cell lines was shown to induce dynamic changes in cell morphology and reorganization of the actin cytoskeleton, with formation of a network of thick stress fibers and assembly of fibronectin fibrils at large focal adhesions. Moreover, NME1 expression results in adhesion reprogramming through an impact on integrin repertoire and focal adhesion dynamics. Having previously demonstrated that NME1 expression promotes repair of DNA damage induced by ultraviolet radiation (UVR) in both yeast and mammalian cells, probably via the nucleotide excision repair pathway, we have more recently demonstrated that NME1 is rapidly recruited to double-strand breaks. This preliminary result represents the first evidence of direct interactions between NME1 and DNA in the context of DNA repair and has set the stage for current efforts to probe its functional interactions with double-strand break repair pathways. Discussed herein are molecular models to explain the interactions of NME1 with such diverse cellular functions as cell motility and DNA repair, potentially through its nucleoside diphosphate kinase and 3'-5' exonuclease activities.

NME1 suppression promotes growth, adhesion and implantation of endometrial stromal cells via Akt and MAPK/Erk1/2 signal pathways in the endometriotic milieu

Is Nometastatic gene 23-H1 (NME1, also known as nm23-H1) involved in regulating the biological behavior of endometrial stromal cells (ESCs), and does it participate in the pathogenesis of endometriosis? NME1 suppression induces ESC dysfunction in the endometriotic milieu. NME1 is a wide-spectrum tumor metastasis suppressor gene that plays an important role in suppressing the invasion and metastasis of tumor cells. An in vitro investigation of the effect of NME1 on the proliferation, adhesion and invasion of eutopic ESCs from patients with endometriosis. Primary ESCs were prepared from 12 samples of ectopic endometrial tissue (6 peritoneal and 6 ovarian lesions), 18 samples of eutopic endometrial tissues (16 from women with ovarian and 2 from women with pelvic endometriomas) and 12 samples of normal endometrial tissue from women without endometriosis, after the tissues had been analyzed histologically. The growth, invasiveness and adhesion of ESCs were studied by the 5-bromo-2'-deoxyuridine cell proliferation assay and by the Matrigel invasion and adhesion assay. Additionally, the effects of NME1 on the activation or expression of related regulatory proteins were investigated by in-cell Western and flow cytometry assays. Expression of NME1 in ESCs derived from eutopic or ectopic endometrium from women with endometriosis is lower than in ESCs from women without endometriosis. Estrogen could down-regulate NME1 expression in ESCs. Silencing NME1 in ESCs promoted the expression of proliferating cell nuclear antigen (PCNA), the anti-apoptotic molecule, survivin, and the adhesion-related molecules, integrin β1 and integrin ανβ3. Silencing NME1 also stimulated ESC proliferation, adhesion and invasion but these effects were inhibited by MAPK/Erk and/or Akt blockers. Further studies are needed to examine the regulatory mechanism of estrogen on NME1 expression of ESCs. Abnormally low expression of NME1 in ESCs may be involved in the pathogenesis of endometriosis by up-regulating growth, adhesion and invasion of ESCs via activating the Akt and MAPK/Erk1/2 signal pathways. This work was supported by National Natural Science Foundation of China (NSFC) (31270969, 31101064 and 81270677) and Program for ZhouXue of Fudan University. None of the authors has any conflict of interest to declare.

Thymic stromal lymphopoietin downregulates NME1 expression and promotes invasion in human trophoblasts via the activation of STAT3 signaling pathway

Our previous study has demonstrated that TSLP induces the invasion of human trophoblasts and plays a role in embryo implantation. The present study is undertaken to explore the mechanism by which TSLP modulates trophoblast invasion. It was revealed that TSLP treatment significantly downregulated NME1 and TIMP1 expression in both human trophoblasts and JEG-3 cells. The stimulatory effect of TSLP on trophoblast invasion was partially inhibited by either STAT3 inhibitor or STAT5 inhibitor. The downregulation of NME1 expression by TSLP was completely abrogated by STAT3 inhibitor. TIMP1 expression was attenuated by NME1 siRNA interference and enhanced by NME1 overexpression in JEG-3 cells. TSLP-stimulated increase in trophoblast invasion was partially attenuated by NME1 overexpression. Our data suggest that TSLP might downregulate NME1 expression via STAT3 signaling pathway, affecting TIMP1 expression in influencing trophoblast invasion. Our studies suggest that further studies on TSLP as a potential therapeutic for recurrent spontaneous abortion are warranted.

Delayed Processing of Bone Marrow Samples Reveals a Prognostic Pattern of NME mRNA Expression in Cytogenetically Normal AML

Abstract 4904 Background:Prognostic molecular markers are expected to be of increasing value in stratifying treatment of patients with AML lacking cytogenetic abnormalities. While specific DNA-level mutations such as those in the FLT-3 or NPM1 genes clearly identify informative genotypes, phenotypic characterization of gene expression should be a more direct indicator of cell function. However, of the prognostic mRNA levels identified to date, such as BAALC, ERG or NME1/2, none appear to be superior to DNA mutation as indicators of prognosis. Because of the lability of mRNA expression, it is common practice to quantify levels only in freshly processed or direct-lysed samples in order to provide a snapshot of gene expression as close as possible to that of fresh tissue. However, characteristics of leukemic cells relevant to prognosis might become more apparent under challenging conditions, such as the stress environment developing in a bone marrow sample during transport or storage. The NME1 and -2 mRNAs have been reported to be prognostic indicators in AML and are multifunctional proteins coordinating metabolism, signalling and gene expression. To see how storage related stress affects the prognostic power of NME mRNA, we have assessed their prognostic relevance in CN-AML bone marrow samples which had originally been processed either directly, or following ≥ 2 days of transport from collaborating centers. Methods:A total of 78 archived CN-AML BMMNC samples were available for this analysis. All samples were taken at presentation from patients under 60ys treated within the AML 96 (OSHO #033) or AML 2002 (OSHO #061) protocols of the East German Study Group (OSHO). Of the 78 samples, 25 (32%) originated locally and had been freshly processed to cryopreserved MNC, while the remaining 53 (68%) had been submitted as bone marrow from other centers with an associated delay of 2–3 days. NME1 and NME2 mRNA levels were determined by triplicate qRT-PCR determinations normalized to RPLP0. The mean NME1 and NME2 expression values from each patient were expressed relative to the corresponding mean NME expression of 17 healthy donor BM samples (control group).To investigate directly the effects of delayed processing on NME mRNA expression, aliquots of 11 fresh CN-AML bone marrow samples were removed for analysis either immediately or following storage at room temperature for ≥ 2 days. Results:Both NME1 and -2 mRNA were increased relative to controls in 84% (21/25) of fresh samples with no prognostic relevance of expression above or below the median. Comparable results were obtained from a cohort of 59 freshly processed CN-AML samples from a separate study. In contrast, the transported samples yielded a heterogeneous pattern of NME mRNA expression above and below control levels, with a significant correlation between NME2 mRNA and event-free survival (p=0.009) independently of FLT-3ITD status. Direct analysis of aliquoted AML bone marrow samples confirmed that a delay in processing of ≥ 2 days resulted in a universal decrease in NME1 mRNA with variable changes in NME2 mRNA. Conclusion:NME1/2 mRNA levels are not indicative of prognosis in freshly processed bone marrow from CN-AML patients <60ys. However, delayed processing is associated with the development of an NME2 expression pattern with high prognostic relevance, maintenance of high NME2 in samples with reduced NME1 being a strong indicator of increased event-free survival. These results suggest that subjecting leukemic cells to defined challenges ex-vivo may reveal phenotypic properties of high relevance to treatment optimization. Disclosures:No relevant conflicts of interest to declare.

Proteomic analysis of NME1/NDPK A null mouse liver: evidence for a post-translational regulation of annexin IV and EF-1Bα

NME/NDPK family proteins are involved in the control of intracellular nucleotide homeostasis as well as in both physiological and pathological cellular processes, such as proliferation, differentiation, development, apoptosis, and metastasis dissemination, through mechanisms still largely unknown. One family member, NME1/NDPK-A, is a metastasis suppressor, yet the primary physiological functions of this protein are still missing. The purpose of this study was to identify new NME1/NDPK-A-dependent biological functions and pathways regulated by this gene in the liver. We analyzed the proteomes of wild-type and transgenic NME1-null mouse livers by combining two-dimensional gel electrophoresis and mass spectrometry (matrix-assisted laser desorption/ionization time of flight and liquid chromatography-tandem mass spectrometry). We found that the levels of three proteins, namely, phenylalanine hydroxylase, annexin IV, and elongation factor 1 Bα (EF-1Bα), were strongly reduced in the cytosolic fraction of NME1(-/-) mouse livers when compared to the wild type. This was confirmed by immunoblotting analysis. No concomitant reduction in the corresponding messenger RNAs or of total protein level was observed, however, suggesting that NME1 controls annexin IV and EF-1Bα amounts by post-translational mechanisms. NME1 deletion induced a change in the subcellular location of annexin IV in hepatocytes resulting in enrichment of this protein at the plasma membrane. We also observed a redistribution of EF-1Bα in NME1(-/-) hepatocytes to an intracytoplasmic compartment that colocalized with a marker of the reticulum endoplasmic. Finally, we found reduced expression of annexin IV coincident with decreased NME1 expression in a panel of different carcinoma cell lines. Taken together, our data suggest for the first time that NME1 might regulate the subcellular trafficking of annexin IV and EF-1Bα. The potential role of these proteins in metastatic dissemination is discussed.

NME1 at the human maternal–fetal interface downregulates titin expression and invasiveness of trophoblast cells via MAPK pathway in early pregnancy

Nometastatic gene 23-H1 (NME1, also known as nm23-H1) is a wide-spectrum tumor metastasis suppressor gene that plays an important role in suppressing the invasion and metastasis of tumor cells. It has been demonstrated that NME1 is also expressed in human first-trimester placenta, but its function at maternal-fetal interface is not clear. The present study aimed to elucidate the biological function of NME1 at the maternal-fetal interface, especially on invasion of the human extravillous cytotrophoblasts (EVCTs). NME1 has been identified in both human trophoblast cells and decidual stromal cells (DSCs) in early pregnancy. We have proved that NME1 silencing in vitro increases the titin protein translation in the invasive EVCTs. Moreover, NME1 can inactivate the phospho-extracellular signal-regulated kinase 1/2 (P-ERK1/2) in trophoblasts in a time-dependent manner, and U0126, an inhibitor of MAPK/ERK, can inhibit partly the enhanced invasiveness and titin expression in trophoblasts induced by NME1 silencing. Interestingly, the expression of NME1 in either villi or decidua is higher significantly in miscarriage than that of the normal early pregnancy. These findings first reveal that the NME1 expressed in trophoblasts and DSCs controls the inappropriate invasion of human first-trimester trophoblast cells via MAPK/ERK1/2 signal pathway, and the overexpression of NME1 at maternal-fetal interface leads to pregnancy wastage.

CDNA array analysis of cytobrush-collected normal and malignant cervical epithelial cells: a feasibility study

Analysis of gene expression pattern is a useful approach to evaluating the biological behavior and clinical outcome of several human malignancies. Differentially expressed genes in malignant squamous cervical cells and the feasibility of gene expression profiling on squamous cervical cells obtained from cervical swabs were investigated. Cervical squamous cells from three women with high-risk human papilloma virus (HR-HPV) positive invasive squamous cervical carcinoma and from three HPV-negative women with normal ectocervical smears were analyzed with cDNA array. Immunoblot analysis was performed to detect the proteins corresponding to the highest upregulated genes with cDNA array. mRNA expression of ERBB2, KIT, FLT1, MYCN, RAS, CDKN2A, CCND1, NME1, NME2, MET, FGF7, FGFR2, and STAT1 was increased in malignant samples. Several expressed genes associated with antiapoptosis (such as BCL2), cell structuring, or cell attachment were also upregulated in carcinoma cells. Decreased gene expression was observed for members of the transforming growth factor receptor superfamily (TGF) and integrin family, interleukin 1 ( IL1), and insulin-like growth factor binding proteins (IGFBPs). This study shows the feasibility of gene expression profiling of cervical squamous cells obtained with cytobrushes by identifying a characteristic gene expression pattern that clearly distinguishes between malignant and normal cervical epithelia of squamous type. We hypothesize that this noninvasive technique could be used in the evaluation of ambiguous Papanicolaou (PAP) smears.

Suppression of human melanoma metastasis following introduction of chromosome 6 is independent of NME1 (Nm23).

Metastasis is suppressed more than 95% following microcell-mediated transfer of a single copy of neomycin-tagged human chromosome 6 (neo6) into the human melanoma cell lines C8161 and MelJuSo. Concomitant with metastasis suppression is upregulation of NME1 (Nm23-H1) mRNA and protein expression. The purposes of this study were to determine whether NME1 expression was responsible for metastasis suppression in neo6/melanoma hybrids, and whether genes on chromosome 6 regulate NME1. Using neo6/C8161 cells, transfection of CAT reporter constructs linked to the NME1 promoter failed to consistently induce CAT. Therefore, it does not appear that genes on chromosome 6 directly control transcription of NME1. Transfection and overexpression of NME1 in MelJuSo, under the control of the CMV promoter, resulted in 40-80% inhibition of lung metastasis following i.v. inoculation of 2 x 10(5) cells. Only one transfectant of C8161 subclone 9 (C8161cl.9) cells was suppressed for metastasis. Control transfections with pCMVneo or pSV2neo did not suppress metastasis in either cell line. Taken together, these data suggest that NME1 can reduce metastatic potential of some human melanoma cells; but, this inhibitory activity appears to be independent of the metastasis suppression following introduction of chromosome 6 into C8161 and MelJuSo human melanoma cell lines.

Potential prognostic value in human breast cancer of cytosolic Nme1 protein detection using an original hen specific antibody.

The metastasis-suppressor nme gene (also called nm23), first identified in murine melanoma cells, exists as two forms in human: nme1 and nme2. However, only the lack of expression of nme1 has been related to distant metastasis appearance in human breast cancer. The aim of this work was first to raise specific antibodies to allow the analysis of Nme1 and then, with this specific tool, to evaluate the predictive value of Nme1 detection in cytosolic extracts of human breast tumours. We obtained a hen antibody that specifically reacts with Nme1 without any cross-reaction with Nme2. We analysed the expression of the protein in 59 human breast tumours and found a significant relationship between this expression and oestrogen receptor status (P<0.001). Moreover, Nme1 expression is related to metastasis-free survival (P<0.001) and survival of patients (P<0.001). The determination of Nme1 expression in primary tumours using our antibody should be an interesting predictive test of the metastasis for clinical investigations.

Increased expression of the NME1 gene is associated with metastasis in epithelial ovarian cancer.

The genetic events involved in the development of metastases of epithelial ovarian cancer are largely unknown. One gene postulated to play a role in tumour metastasis suppression is NME1 (nm23-H1), and an inverse relationship between NME1 expression and metastatic potential has been observed for some solid tumours. In this study we have investigated the levels of mRNA expression of the 2 isoforms of the NME gene, NME1 and NME2. A maximum of 45 tumours samples from 33 patients were available for Northern blot analysis. We observed variable levels expression of NME1 and NME2 mRNA. The average level of NME1, but not NME2, mRNA expression was statistically higher in metastatic biopsies when compared with primary tumour biopsies. To examine the possible tumour suppressor gene role of NME1 in ovarian tumours, 76 patients were investigated by Southern blot analysis to determine the rate of allelic deletion. Allele loss at 5 other chromosome 17 loci (D17S5, TP53, NF1, D17S74, D17S4) was also evaluated for many of these 76 patients. Allele loss was observed in 22/30 (73%) informative patients at the NME1 locus. We also observed high rates of allele loss at the other loci evaluated. No correlations with clinical stage, histological subtype or patient survival were observed in either mRNA or DNA analyses. We have established that tumour progression in ovarian cancer is accompanied by over-expression of the NME1 gene; however, despite high rates of allele loss at the NME1 locus, the concept that NME1 may be a candidate tumour suppressor gene in ovarian cancer cannot be confirmed by this study.

Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae.

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype. Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.